The present technical ability to sequence entire mammalian genomes outpaces current computational and experimental abilities to decode the information contained within these sequences. Cross-species sequence comparisons are a powerful method for decoding genomic information due to the fact that functional elements are conserved through evolution whereas nonfunctional sequences drift. Because the rate of evolution varies widely in different regions of a genome within a species as well as for orthologous sequences between species, a comprehensive comparative analysis of the human genome will require that it be compared with the genomic sequences of multiple mammals. At the current cost of sequencing mammalian genomes it is too expensive to obtain in-depth sequence coverage of more than a few mammals. Thus, we are left with the choice of forgoing the information that would be obtained by multi-species comparisons or employing a high-resolution scanning approach, such as described in this proposal, for comparing human sequences with the DNA of multiple mammals. In phase I of the proposed research, we will analyze the data from a pilot study in which a 300-kb region of human chromosome 21 has been compared with the DNA of five mammals using oligonucleotide high-density arrays. We will improve the current algorithms employed for detecting and characterizing evolutionarily conserved sequences using high-density arrays. In addition, we will demonstrate that multi-species comparative analysis using high-density arrays combined with targeted dideoxy-sequencing can obtain the homologous sequences of biologically interesting conserved elements across multiple mammals. During phase II of this proposal, we will screen approximately 32 Mb of human chromosome 21 for conserved sequences by hybridizing high-density arrays with orthologous dog, cat, cow, pig, and horse DNA. The goal of this proposal is to demonstrate the feasibility and cost-effectiveness of the high-density array approach for identifying conserved human elements by comparative analysis with multiple mammals across the entire human genome.